One of the primary causes for untimely loss of organ transplants is the inability to recognize immune injury from acute rejection early, resulting in considerable injury and irreversible loss of function of the organ. With the increasing gap between available organs and patients with end stage kidney disease awaiting organ transplants, and the only mechanism of definitely classifying organ injury being dependent on an invasive biopsy, improved methods for early and non-invasive detection of transplant injury is a critical unmet need. This is an application for renewal of an existing RO1, which conducted human urine proteomic studies by sophisticated mass spectrometric and bioinformatic techniques in 396 pediatric and adult kidney transplant recipients, and resulting in the identification of a 33 urine protein panel that classifies acute rejection, chronic rejection and vral inflammation from BK virus infection, with >80% accuracy by a non-invasive urine protein assay. With expertise in clinical trial design, urine sample acquisition and processing from multiple clinical sites, clinical database design, access to archived, highly annotated samples from 600 kidney transplant recipients, access to serial urine samples from two randomized clinical trials and deep expertise in biostatistics, this mature team of investigators now propose to use these selected proteins to generate a transplant injury atlas in the renal transplant biopsy by a novel multiplex immunofluorescence/ in situ hybridization (miFish) assay (Aim 1) and to interrogate the clinical utility of the 33 urine proteins by a customized multiplex MSD- ELISA assay (Aim 2) for prediction of rejection and infection specific transplant injury, accurate staging of rejection and to evaluate these biomarkers as surrogate outcome measures of therapy. The benefit of this research would be to move urine protein non-invasive markers for clinical evaluation of graft rejection ready for out-patient clinical application avoid biopsies and individualize immunosuppression, while avoiding rejection, thus supporting the application of predictive, personalized and precision medicine for the kidney transplant recipient.